CCN1 expression in hepatocytes contributes to macrophage infiltration in nonalcoholic fatty liver disease in mice

Zhaolian Bian; Yanshen Peng; Zhengrui You; Qixia Wang; Qi Miao; Yuan Liu; Xiaofeng Han; Dekai Qiu; Zhiping Li; Xiong Ma

doi:10.1194/jlr.M026013

. 2013 Jan;54(1):44–54. doi: 10.1194/jlr.M026013

CCN1 expression in hepatocytes contributes to macrophage infiltration in nonalcoholic fatty liver disease in mice^[S]

Zhaolian Bian ^*,^†,¹, Yanshen Peng ^*,^†,¹, Zhengrui You ^*,^†,¹, Qixia Wang ^*,^†, Qi Miao ^*,^†, Yuan Liu ^*,^†, Xiaofeng Han ^*,^†, Dekai Qiu ^*,^†, Zhiping Li ^§, Xiong Ma ^*,^†,²

PMCID: PMC3520538 PMID: 23071295

Abstract

Our objective was to investigate the potential roles of CCN1 in the inflammation and macrophage infiltration of nonalcoholic fatty liver disease (NAFLD). The regulation of hepatic CCN1 expression was investigated in vitro with murine primary hepatocytes treated with free fatty acids or lipopolysaccharide (LPS) and in vivo with high-fat (HF) diet-fed mice or ob/ob mice. CCN1 protein and a liver-specific CCN1 expression plasmid were administered to mice fed a normal diet (ND) or HF diet. Myeloid-derived macrophages and RAW264.7 cells were also treated with CCN1 in vitro to determine the chemotactic effects of CCN1 on macrophages. LPS treatment significantly increased hepatic CCN1 expression in HF diet-fed mice and ob/ob mice. LPS and FFAs induced CCN1 expression in primary murine hepatocytes in vitro through the TLR4/MyD88/AP-1 pathway. CCN1 protein and overexpression of CCN1 in the liver induced more severe hepatic inflammation and macrophage infiltrates in HF mice than in ND mice. CCN1 recruited macrophages through activation of the Mek/Erk signaling pathway in myeloid-derived macrophages and RAW264.7 cells in vitro. Endotoxin and FFA-induced CCN1 expression in hepatocytes is involved in the hepatic proinflammatory response and macrophage infiltration in murine NAFLD.

Keywords: cell signaling, cytokines, diet and dietary lipids, fatty acid, Cyr61, chemotaxis, nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) begins with the aberrant accumulation of triglycerides in the liver (simple steatosis), which in some individuals elicits an inflammatory response (nonalcoholic steatohepatitis, NASH) that can progress to cirrhosis and liver cancer (1). NASH is characterized by hepatocellular ballooning, lobular inflammation, and fibrosis in histology (2–4). A good model to explain the pathogenesis of NASH is the “multiple parallel hits” hypothesis proposed by Tilg and Moschen, which states that various parallel processes, especially gut-derived and adipose tissue-derived factors, may be conducive to the development of liver inflammation in NAFLD (5).

Members of the CCN (Cyr61/CTGF/NOV) family have emerged as dynamically expressed, extracellular matrix-associated proteins that play critical roles in regulating cell adhesion, migration, proliferation, differentiation, apoptosis, and survival (6, 7). CCN1 (also known as cysteine-rich protein 61, Cyr61) is normally expressed at a low level in most tissues but is increased as a result of inflammation or tissue repair. It has been shown that CCN1 activates the NF-κB signaling pathway in macrophages, leading to the expression of multiple proinflammatory cytokines and chemokines characteristic of classical activated M1 macrophages (8). The definitive role of CCN1 in liver diseases remains unclear, although it has been shown to be a tumor suppressor in hepatocarcinogenesis (9) and is involved in the oxidative DNA damage response in ConA-treated livers (10). In this study, we showed for the first time that induction of CCN1 by FFAs and LPS in hepatocytes resulted in macrophage infiltration and inflammation in the liver in hepatic steatosis.

MATERIALS AND METHODS

Animal and treatments

Male C57BL/6 mice (6–8 weeks of age) were purchased from the Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China) and housed under pathogen-free conditions in the animal facility of the Shanghai Jiao Tong University School of Medicine. Mice were fed either a high-fat (HF) diet (SLAC Laboratory Animal Co. Ltd) providing 59% of calories from fat, 25% from carbohydrates, and 16% from protein for eight weeks or an isocaloric normal diet (ND) containing less fat (12% fat, 59% total carbohydrate. and 29% protein). Male C57BL/10ScN toll-like receptor 4 (TLR4) knockout mice, male C57BL/6 myeloid differentiation factor 88 (MyD88) knockout mice, their control littermates, and male C57BL/6 ob/ob mice (6–8 weeks of age) were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). All mice were maintained in a temperature- and light-controlled facility with ad libitum access to food and water.

Escherichia coli LPS (50 μg/mouse, Sigma, St. Louis, MO) was injected intraperitoneally, and mice were euthanized 6 h later. The CCN1 protein displays a remarkable degree of evolutionary conservation, with 92.8% identity between mouse and human CCN1 (11). Recombinant human CCN1 (5 μg/mouse, R and D Systems, Minneapolis, MN) was administered intravenously through the tail vein, and mice were euthanized 24 h later. The neutralizing anti-integrin α_M antibody (0.1 mg/mouse, BD Pharmingen, San Diego, CA) or Z-VAD-FMK (200 μg/mouse, Sigma), which inhibits induction of apoptosis, was injected through the tail vein 2 h before administration of CCN1. Control animals were injected with vehicle only.

The plasmid encoding mouse CCN1 cDNA or control vector (50 μg/mouse) was dissolved in PBS with a volume of 2 ml or 3 ml and then delivered to ND and HF mice by hydrodynamic tail vein injection, respectively (12). All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine and were conducted in accordance with the National Research Council Guide for Care and Use of Laboratory Animals.

Liver-specific CNN1 expression plasmid construction

The construction of the liver-specific CCN1 expression plasmid was carried out as previously described (12), using the Enh1mTTR (ET) promoter generated by fusing a synthetic hepatocyte-specific enhancer to the murine transthyretin promoter (13). The CCN1 cDNA inserted into the vector was expressed under the control of ET promoter. The control plasmid contained the GFP gene under control of the same promoter.

Cell culture

Primary murine hepatocytes were isolated from mice by in situ collagenase liver perfusion (14) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 mg/ml streptomycin, and 100 U/ml penicillin (Invitrogen, Carlsbad, CA).

Primary murine macrophages were obtained as previously described with minor modifications (15) and cultured in DMEM supplemented with 10% FBS, 20 ng/ml macrophage colony-stimulating factor (M-CSF; R and D Systems), 100 mg/ml streptomycin, and 100 U/ml penicillin. The macrophage RAW264.7 cell line was purchased from the Shanghai Institute of Cell Biology (Chinese Academy of Sciences, Shanghai, China) and cultured with DMEM plus 10% FBS.

Cells were precultured with JNK inhibitor II (SP600125, 10 μM), IKK-2 inhibitor V (IMD0354, 1 μM), U0126 (10 μM), PD98059 (10 μM) (Merck, Darmstadt, Germany), or anti-integrin α_M (5 μg/ml, BD Pharmingen) for 2 h before stimulation with LPS, OP 2:1 (mixture of oleic acid and palmitic acid at a ratio of 2:1, Sigma) or CCN1, respectively.

Chemotaxis assay

RAW264.7 cells or murine myeloid-derived macrophages were precultured with or without neutralizing anti-integrin α_M (5 μg/ml), U0126 (10 μM) for 30 min, and then 1 × 10⁵ cells were placed on a 0.8 μm membrane (Millicell Hanging Cell Culture Insert, PET, Millipore, Billerica, MA) in DMEM with 10% FBS. The cells were allowed to migrate toward CCN1 in the medium below the membrane at the indicated concentrations (250 ng/ml, 500 ng/ml, 1,000 ng/ml) for 90 min and 24 h, respectively. Migration was quantified as the number of crystal violet-stained cells observed on the underside of the membrane by light microscopy (10 randomly chosen fields per membrane and three replicate wells per treatment).

Western blot

Immunoblotting analyses were carried out using either cell extracts or whole-liver extracts, as described previously (16). Immunoblots were probed with antibodies to CCN1 (Abcam, Cambridge, UK), phosphorylated and total Mek1/2, Erk1/2, C-jun, and IκB-α (Cell Signaling Technology, Boston, MA).

Immunohistochemistry and TUNEL assay

Immunohistochemistry was performed on formalin-fixed, paraffin-embedded liver tissues using antibodies against F4/80 (1:100, Genetex, Irvine, CA) and CCN1 (1:100). The procedures were performed as previously described with minor modifications (17). Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), using the TUNEL FITC kit (ShanghaRuian bioTechnologies Co., Ltd, Shanghai, China) according to the manufacturer's instructions.

Statistical analysis

Data are expressed as the mean ± SD. The group means were compared using ANOVA. All statistical analysis was performed using the SPSS statistical software version 16.0 (SPSS Inc., Chicago, IL). P values < 0.05 were considered statistically significant.

RESULTS

LPS increases hepatic CCN1 expression in murine NAFLD

Our previous studies demonstrated that mice fed HF diets became obese and developed hepatic steatosis (18, 19). In the present study, HF mice also developed significant hepatic steatosis characterized by the presence of macrovesicular and microvesicular lipid droplets. Immunohistochemical analysis showed that LPS administration significantly increased hepatic CCN1 expression in HF mice compared with HF mice without LPS administration and LPS-treated ND mice (Fig. 1A, upper panel). To confirm that hepatic CCN1 was increased in different steatosis models, ob/ob mice were treated with or without LPS. Similarly, hepatic CCN1 expression was increased in ob/ob mice compared with WT mice. LPS treatment further increased hepatic CCN1 expression in ob/ob mice (Fig. 1A, lower panel).

Fig. 1. — LPS increases hepatic CCN1 expression in murine NAFLD. (A) Representative immunohistochemical staining for hepatic CCN1 expression in ND and HF mice and in WT and ob/ob mice, treated with or without LPS. (B) Representative Western blot analysis of hepatic CCN1 expression in ND and HF mice treated with or without LPS. (C) Representative Western blot analysis of hepatic CCN1 expression in ob/ob mice treated with or without LPS. (D) Representative Western blot analysis of CCN1 expression in murine hepatocytes (MHC) and liver nonparenchymal cells (LNPC). (n = 5 in each group, *P < 0.05; **P < 0.01).

Western blot analysis showed that both HF and LPS treatment increased hepatic expression of CCN1 in ND mice. However, LPS-treated HF mice had significantly higher hepatic CCN1 expression compared with the LPS-treated ND mice (Fig. 1B). The same phenomenon was also observed in ob/ob mice (Fig. 1C). By immunohistochemical staining analysis (Fig. 1A), we found that CCN1 was mainly expressed in hepatocytes. Similarly, Western blot analysis showed that the hepatocytes had significantly higher CCN1 expression than the nonparenchymal cells, suggesting that the major source of CCN1 protein in the liver is hepatocytes (Fig. 1D). Therefore, we chose hepatocytes as target cells for our next experiments to explore the regulation of CCN1 expression.

LPS and FFAs induce CCN1 expression in primary murine hepatocytes through the TLR4/MyD88/AP-1 signaling pathway

LPS at the concentrations of 1 μg/ml and 2 μg/ml induced CCN1 expression by approximately 3-fold in primary murine hepatocytes in vitro (Fig. 2A, upper panel). In addition, CCN1 expression was detectable at 12 h and reached a maximum value at 48 h after LPS treatment at 2 μg/ml (Fig. 2A, lower panel). Primary murine hepatocytes were incubated with a mixture of long chain fatty acids (OP 2:1) to establish a cellular steatosis model (20). Similarly, FFAs (OP 2:1) also induced CCN1 expression in a concentration-dependent manner, peaking at 0.5 mmol/l (Fig. 2B, upper panel). At 0.5 mmol/l, FFAs were shown to induce the maximum CCN1 expression level at 24 h (Fig. 2B, lower panel). These results demonstrated that LPS and FFAs both induced CCN1 expression of hepatocytes in a time- and concentration-dependent manner.

In vivo, LPS administration nearly tripled the hepatic CCN1 expression in WT mice; however, no significant difference in CCN1 expression was detected between treatment with LPS and treatment without LPS in TLR4^−/−and MyD88^−/−mice (Fig. 3A). Similarly, LPS and FFAs could not induce CCN1 expression in hepatocytes isolated from TLR4^−/−or MyD88^−/−mice (Fig. 3B). These results suggest that LPS and FFA-induced CCN1 expression is dependent on the TLR4/MyD88 signaling pathway.

Fig. 3. — LPS and FFAs induce CCN1 expression in primary murine hepatocytes through the TLR4/MyD88/AP-1 signaling pathway. (A) Representative Western blot analysis of hepatic CCN1 expression in WT and TLR4^−/−mice or MyD88^−/−mice treated with or without LPS. (B) Representative Western blot analysis of CCN1 expression in murine primary hepatocytes from WT, TLR4^−/−, and MyD88^−/−mice, treated with or without LPS and OP 2:1. (C) Representative Western blot analysis of CCN1 expression in murine primary hepatocytes treated with LPS, OP 2:1, SP600125, and IMD0354. (n = 3 in each group, *P < 0.05, **P < 0.01).

To illustrate the downstream signaling pathway of TLR4/MyD88 involved in this process, inhibitors for the AP-1 (SP600125) and NF-κB (IMD0354) pathways were used. SP600125 and IMD0354 significantly inhibited the phosphorylations of c-Jun (component of AP-1) and IKBα, respectively (supplementary Fig. I-A). However, only SP600125 inhibited the expression of CCN1 in primary hepatocytes stimulated with LPS or FFAs (Fig. 3C). These results suggest that LPS and FFAs induce CCN1 expression in hepatocytes through activation of the TLR4/MyD88/AP-1 signaling pathway.

CCN1 induces severe hepatic inflammation and liver injury in murine NAFLD

CCN1 protein induced mild to moderate hepatic inflammation in ND mice, whereas it induced severe hepatic inflammation in HF mice (Fig. 4A). CCN1 treatment increased the numbers of hepatic inflammatory foci in both ND and HF mice compared with ND and HF mice without treatment, respectively. Furthermore, CCN1 induced more hepatic inflammatory foci in HF mice compared with ND mice treated with CCN1 (Fig. 4B). Similarly, CCN1 treatment increased serum ALT and AST levels in both ND and HF mice (Fig. 4C, D). Consistent with the histological changes, CCN1-treated HF mice had higher ALT levels than CCN1-treated ND mice (Fig. 4C), suggesting that the HF mice were more susceptible to CCN1-induced hepatic inflammation and liver injury.

As CCN1 induces macrophage adhesion and activation through integrin α_Mβ₂ (8, 21), a neutralizing antibody against integrin α_M was used to block the binding between CCN1 and macrophages. Neutralization of integrin α_M attenuated CCN1-induced hepatic inflammation in ND and HF mice, indicated by less hepatic inflammatory infiltration in histological examinations and lower serum ALT and AST levels compared with ND and HF mice treated with CCN1 only (Fig. 4A–D). These results suggested that CCN1 induced monocyte infiltration through binding to integrin α_M on the surface of monocytes. CCN1 also induced moderate hepatic inflammation in ob/ob mice but not to the extent seen in HF mice (supplementary Fig. I-B). Delivery of a liver-specific CCN1 plasmid, confirmed to successfully overexpress CCN1 (supplementary Fig. I-C), induced more severe hepatic inflammation in HF mice than in ND mice (Fig. 4E).

CCN1 induces macrophage infiltration through binding to integrin α_M in vivo

Next, we analyzed macrophage infiltration in CCN1 protein-induced hepatic inflammatory foci in both ND and HF mice by using F4/80 immunohistochemistry. F4/80 positive cells were frequently detected (Fig. 5A), suggesting that macrophages are the major cellular type infiltrated in CCN1-induced liver injury. More importantly, CCN1 induced more macrophage infiltrates in HF mice compared with ND mice treated with CCN1 (Fig. 5B). Accordingly, neutralization of integrin α_M decreased CCN1-induced hepatic macrophage infiltrates in ND and HF mice (Fig. 5A, B). Flow cytometric analysis showed that the majority of F4/80 positive cells in the liver expressed integrin α_M (Fig. 5C). These results suggest that integrin α_M may mediate macrophage infiltration in CCN1-induced hepatic inflammation. Consistent with the results of CCN1 protein administration, the forced expression of CCN1 also induced numerous macrophage infiltrations in both ND and HF mice (Fig. 5D).

Fig. 5. — CCN1 induces macrophage infiltration in murine NAFLD. (A) Representative immunohistochemical stain of F4/80 in the livers (magnification 400×) of ND and HF mice that were either untreated or treated with CCN1 protein with or without neutralizing antibody against integrin α_M. (B) Numbers of F4/80 positive cells per 40 × 10 field in liver tissues obtained from mice that were either untreated or treated with CCN1 protein with or without neutralizing antibody against integrin α_M. (C) Flow cytometric analysis of F4/80 and integrin α_M expression in monocytes (gated on CD45 positive cells) from murine livers. (D) Representative immunohistochemical stain of F4/80 in liver sections (magnification 200×) from ND and HF mice with forced CCN1 expression. (n = 5 in each group, *P < 0.05, **P < 0.01).

CCN1-induced hepatic inflammation and macrophage infiltration are not affected by apoptosis inhibitor in vivo

It was reported that CCN1 could facilitate apoptosis mediated by Fas and TNF-α in vivo and vitro (10, 22). Therefore, we observed whether cellular apoptosis played a critical role in hepatic inflammation and macrophage infiltration induced by CCN1. Indeed, CCN1 induced cellular apoptosis in the livers not only of ND mice but also of HF mice, and Z-VAD-FMK, a caspase inhibitor, restrained this process (Fig. 6A). However, Z-VAD-FMK did not significantly attenuate the hepatic inflammation induced by CCN1 in either ND or HF mice (Fig. 6B). In addition, CCN1-induced macrophage infiltration was not significantly reduced by Z-VAD-FMK (Fig. 6C), indicating that the role of CCN1 on hepatic inflammation and macrophage infiltration is independent of cellular apoptosis potentially induced by CCN1.

Fig. 6. — Hepatic inflammation and macrophage infiltration induced by CCN1 are not affected by apoptosis inhibitor in vivo. (A) Representative TUNEL staining of liver sections (magnification 200×) from ND and HF mice that were either untreated or treated with CCN1 protein with or without Z-VAD-FMK. (B) Representative H and E staining of liver sections (magnification 200×) from ND and HF mice that were either untreated or treated with CCN1 protein with or without Z-VAD-FMK. (C) Representative immunohistochemical stain of F4/80 in the livers (magnification 200×) of ND and HF mice that were either untreated or treated with CCN1 protein with or without Z-VAD-FMK.

CCN1 induces macrophage chemotaxis through the integrin α_M/Mek1/2/Erk1/2 signaling pathway in vitro

CCN1 in different dosages increased the phosphorylation of Mek1/2 and Erk1/2 in the RAW264.7 cell line (Fig. 7A). CCN1 recruited primary murine myeloid-derived macrophages in a concentration-dependent manner, ranging from 250 to 1,000 ng/ml both at 90 min and 24 h. Pretreatment with the neutralizing antibody against integrin α_M or an inhibitor for Mek1/2 (U0126) significantly reduced the level of macrophage migration in response to CCN1 (Fig. 7B). Similar results were also found in the murine macrophage RAW264.7 cell line (Fig. 7C). Taken together, these results suggest that CCN1 recruits macrophages through activation of the integrin α_M/Mek1/2/Erk1/2 signaling pathway.

DISCUSSION

In this study, we demonstrated for the first time that FFAs and LPS induced CCN1 expression in hepatocytes through the TLR4/MyD88/AP-1 signaling pathway in vitro and in vivo. CCN1 could induce severe hepatic inflammation and apoptosis in mice with NAFLD, and CCN1 was shown to induce macrophage chemotaxis through the integrin α_M/Mek1/2/Erk1/2 signaling pathway. Therefore, CCN1 may play roles in the inflammatory response and macrophage infiltration in NAFLD.

Accumulating evidence shows that innate immunity is actively involved in insulin resistance and plays a critical role in the pathogenesis of NASH (23). TLR4 links innate immunity and fatty acid-induced insulin resistance (24, 25). TLR4 is a high-affinity receptor for LPS, a component of the cellular wall of gram-negative bacteria, and is considered a pivotal exogenous danger-signaling molecule in the pathogenesis of NAFLD (26). LPS has been shown to induce production of TNF-α in macrophages, thereby triggering TNF-α-induced hepatocyte apoptosis in a murine NASH model (27). In addition, TLR4 plays an important role in mediating proinflammatory effects of saturated FFAs. Saturated FFAs may function as the endogenous substances that contribute to TLR4 activation in the setting of obesity (28). FFAs may elicit hepatotoxicity and stimulate progression from simple steatosis to NASH via several mechanisms beyond direct cytotoxicity (29). Palmitic acid can bind to TLR4, leading to activation of NF-κB and upregulate its target genes, such as TNF-α and IL-6, in macrophages and adipocytes. TLR4-deficient mice develop obesity when fed a HF diet; however, they are still partially protected against development of insulin resistance, possibly due to reduced inflammatory gene expression in liver and fat tissues (24). FFAs also induce production of proinflammatory cytokines TNF-α (30) and IL-8 (31) from hepatocytes, potentially contributing to hepatic inflammation and consequent liver injury. Most recently, Csak and colleagues showed that the saturated FFA palmitic acid upregulates the inflammasome, which cleaves pro-interleukin-1β (pro-IL-1β) into secreted IL-1β, and induces sensitization to LPS for IL-1β release in hepatocytes (32). Our study showed that LPS and FFAs induced production of CCN1 in hepatocytes, which may be another important inflammatory factor in the liver. Therefore, our study and other studies suggest that hepatocytes are not only passively injured targets but that they also are actively involved in orchestrating responses to insults in NASH.

Macrophages are another important component of innate immunity in NAFLD. In adipose tissue, macrophages are a major source of proinflammatory cytokines, which can function in a paracrine and potentially an endocrine fashion to cause decreased insulin sensitivity. The accumulating evidence indicates that activation of hepatic macrophages (Kupffer cells) positioned at the “frontline” is an essential element in the pathogenesis of NAFLD (25). It was shown that CCN1 can activate a proinflammatory genetic program in macrophages, such as monocyte chemotactic protein 1 (MCP-1) and macrophage inflammatory protein 1 α (MIP-1α) (8). In our study, we showed that CCN1 could recruit macrophages through binding to integrin α_M on the cellular surface of macrophages, which at least partially explained the massive macrophage infiltrates in murine liver after CCN1 administration. Therefore, production of CCN1 by hepatocytes after treatment with LPS and FFAs may activate and recruit macrophages into the liver. These activated hepatic macrophages then release various chemokines, such as MCP-1, which in turn recruit additional macrophages, setting up a feed-forward process that further increases the number of macrophages in the liver and propagates the chronic inflammatory state (28). In other words, the proinflammatory process is initiated in NAFLD by CCN1-mediated induction of macrophage infiltration into the liver through direct chemotaxis and secondary recruitment by chemokines.

In summary, we propose for the first time that endotoxins and FFAs induce expression of the matricellular signaling molecule CCN1 in hepatocytes through activation of the TLR4/MyD88/AP-1 signaling pathway in mice. CCN1 then drives the recruitment of macrophages into the steatotic liver, inducing the inflammatory process and macrophage infiltration. The potential role of CCN1 in the process of transition from simple steatosis to NASH in humans will be worth exploring in future studies. Thus, CCN1 may become a potential therapeutic target to prevent disease progression to advanced stages in NAFLD.

Supplementary Material

supplementary Fig.1

supp_54_1_44__index.html^{(986B, html)}

Acknowledgments

The authors thank Xin Liu (Model Animal Research Center of Nanjing University) for his assistance with breeding of TLR4^−/−and MyD88^−/−mice.

Footnotes

Abbreviations:

ALT: alanine aminotransferase
AP-1: activating protein-1
AST: aspartate aminotransferase
CCN1: cysteine-rich protein 61, Cyr61
HF: high-fat
IL: interleukin
LPS: lipopolysaccharide
MCP-1: monocyte chemotactic protein 1
MIP-1α: macrophage inflammatory protein 1 α
MyD88: myeloid differentiation factor 88
NAFLD: nonalcoholic fatty liver disease
NASH: nonalcoholic steatohepatitis
ND: normal diet
TLR: toll-like receptor
TNF-α: tumor necrosis factor α
TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling
WT: wild-type

This work was supported by awards from the National Natural Science Foundation of China (#30770963, #30972751, and #81170380 to X.M.), the Shanghai Pujiang Program, and the Program for Innovative Research Team of Shanghai Municipal Education Commission (to X.M.).

^[S]

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of one figure.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplementary Fig.1

supp_54_1_44__index.html^{(986B, html)}

supp_M026013_jlr.M026013-1.pdf^{(272.6KB, pdf)}

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PERMALINK

CCN1 expression in hepatocytes contributes to macrophage infiltration in nonalcoholic fatty liver disease in mice[S]

Zhaolian Bian

Yanshen Peng

Zhengrui You

Qixia Wang

Qi Miao

Yuan Liu

Xiaofeng Han

Dekai Qiu

Zhiping Li

Xiong Ma

Abstract

MATERIALS AND METHODS

Animal and treatments

Liver-specific CNN1 expression plasmid construction

Cell culture

Chemotaxis assay

Western blot

Immunohistochemistry and TUNEL assay

Statistical analysis

RESULTS

LPS increases hepatic CCN1 expression in murine NAFLD

Fig. 1.

LPS and FFAs induce CCN1 expression in primary murine hepatocytes through the TLR4/MyD88/AP-1 signaling pathway

Fig. 2.

Fig. 3.

CCN1 induces severe hepatic inflammation and liver injury in murine NAFLD

Fig. 4.

CCN1 induces macrophage infiltration through binding to integrin αM in vivo

Fig. 5.

CCN1-induced hepatic inflammation and macrophage infiltration are not affected by apoptosis inhibitor in vivo

Fig. 6.

CCN1 induces macrophage chemotaxis through the integrin αM/Mek1/2/Erk1/2 signaling pathway in vitro

Fig. 7.